Cyclic dodecapeptide and process for the preparation thereof

ABSTRACT

The present invention relates to a novel organochemical compound, referred to as omphalotin for short, a process for its preparation by an essentially microbiological route and its use as microbicide and pesticide, preferably for controlling animal pests, harmful fungi and bacteria.

[0001] The present invention relates to a novel organochemical compound,hereinafter referred to as omphalotin, a process for its preparation byan essentially microbiological route and its use as microbicide andpesticide, preferably for controlling animal pests, harmful fungi andbacteria.

[0002] The novel omphalotin has been found and, based on the presentspectroscopic and other analytical data, the following formula (I) isproposed:

[0003] where—(terminal) represents —CH₃,

[0004] represents

[0005] or (terminal) represents

[0006] represents —CH₂—,

[0007] represents

[0008] and

[0009] represents

[0010] Furthermore, it has been found that the novel omphalotin can beemployed for controlling pests and parasites in plants and warm-bloodedanimals. In particular, it has high activity against nematodes andarthropods (like insects and arachnids), and against microbial pests, inparticular against fungi and bacteria. Owing to these properties, thenovel compound and compositions comprising this compound can be employedparticularly advantageously in crop protection, in the protection ofstored products, in the hygiene sector and in animal breeding and animalhusbandry.

[0011] The novel compound of the formula (I) is obtained by cultivatingsuitable microorganisms from the class of the Basidiomycetes, preferablyfrom the genera Omphalotus and Lampteromyces, particularly preferablyOmphalotus, in a customary manner in a cultured medium containingassimilable carbon and nitrogen sources and mineral salts, under aerobicconditions, and isolating the desired compound by customary methods.

[0012] Knowing the properties of the novel compound according to theinvention, it is possible in an easy and quick manner to select theappropriate microorganism strains which produce the omphalotin accordingto the invention by routine processes, with the aid of customarychromatographic, spectroscopic, microbiological (for example inhibitionzone test) and/or biological methods (for example by determining theactivity against nematodes or insects).

[0013] For the microbiological preparation of the compound according tothe invention, preference is given to using omphalotus strains, inparticular Omphalotus olearius strains (synonym: Clitocybe illudensstrains). Very particular preference is given to using the Omphalotusolearius strains No. 83 039, 90 173, 91 050, 92 095, 93 162 and 90 170and those variants and mutants of these strains which have featureswhich are essential for carrying out the present invention, or whichhave the same function.

[0014] The novel strains can be described as follows: 1. Comparison ofthe morphology of the mycel of various Omphalotus olearius strains onsolid medium (M1) Fungus strain Mycel consistency and colour Omphalotusolearius The mycel which is white at the beginning of growth 83 039 (DSM9737) only changes colour to a pale yellow, in rare cases somewhatbrownish. Secretions, as observed with the other strains, were notobserved. Omphalotus olearius The young mycel is white-fluffy, the oldermycel changes 90 170 (DSM 9742) colour to orange-black. In some casesdroplets of brown colour are secreted. Omphalotus olearius The youngwhite mycel grows less dense and fluffy than 90 173 (DSM 9738) 90 170.The older mycel is initially pale yellow and later changes colour toorange-black. In some cases, brown secretions are observed. Omphalotusolearius Growth and mycel colour are similar to that of 90 170, 91 050(DSM 9739) however, the secretions of brown droplets are more numerous.Omphalotus olearius The young white mycel changes colour to an intense92 095 (DSM 9740) orange, later slightly brownish. The secretion ofbrown droplets is very pronounced. The mycel does not turn black.Omphalotus olearius Similar to 92 095, the young yellow mycel changes 93162 (DSM 9741) colour to an intense orange and likewise secretes manybrown drops.

[0015] 2. Growth of various Omphalotus olearius strains at differenttemperatures in 1 l of a shaken culture M1 medium in a 2 l Erlenmeyerflask Incubation temperature [° C.] Fungus strain 24 27 30 Omphalotusolearius maximum medium strongly 83 039 (DSM 9737) growth rate growthrate reduced growth Omphalotus olearius Slightly less growth thanmaximum 90 170 (DSM 9742) at 30° C. growth rate Omphalotus olearius 90Identical growth at all temperatures 173 (DSM 9738) Omphalotus olearius91 Identical growth at all temperatures 050 (DSM 9739) Omphalotusolearius 92 medium growth maximum lowest 095 (DSM 9740) rate growth rategrowth rate Omphalotus olearius 93 maximum maximum lowest 162 (DSM 9741)growth rate growth rate growth rate

[0016] 3. Form and size of the pellets of the various Omphalotusolearius strains at growth in liquid culture (M1) Fungus strain Form andsize of pellet Omphalotus olearius The fungus forms firm pellets of adiameter of 83 039 (DSM 9737) 0.5 to 1.5 cm Omphalotus olearius Thefungus grows in very small firm pellets 90 170 (DSM 9742) having adiameter of about 0.5 cm Omphalotus olearius The fungus grows with asimilar pellet form and 90 173 (DSM 9738) size as 90 170 Omphalotusolearius The pellet size varies frequently but is generally 91 050 (DSM9739) slightly greater than for 90 170 (0.5 to 1.5 cm). The pellet is“frazzled”. Omphalotus olearius The fungus forms the largest pellets ofa 92 095 (DSM 9740) diameter of 1 to 2 cm and a “prickly” surface.Omphalotus olearius The fungus forms pellets having a diameter of 93 163(DSM 9741) up to 1.5 cm and less firm, more “frazzled” structure.

[0017] The abovementioned Omphalotus olearius strains are novel. Theyhave been deposited at the Deutschen Sammlung von Mikroorganismen undZellkullturen GmbH (DSM), Mascheroder Weg 1b, D 38124 Braunschweig,Federal Republic of Germany, in accordance with the provisions of theTreaty of Budapest on the international recognition of the deposition ofmicroorganisms for the purpose of patent processes and have thefollowing deposition numbers or entry numbers: Strain Number and date ofdeposition Omphalotus olearius No. 83 039 DSM 9737 13.02.1995 Omphalotusolearius No. 90 173 DSM 9738 13.02.1995 Omphalotus olearius No. 91 050DSM 9739 13.02.1995 Omphalotus olearius No. 92 095 DSM 9740 13.02.1995Omphalotus olearius No. 93 162 DSM 9741 13.02.1995 Omphalotus oleariusNo. 90 179 DSM 9742 13.02.1995

[0018] The structure of the Omphalotus isolated from Omphalotus oleariuswas elucidated with the aid of ¹H, ¹³C, COSY, NOESY, HMQC and HMBC NMRspectra, and an ESI MS spectrum. The molecular mass was determined to be1318 Da. The NMR spectra were recorded using a solution of about 3 mg ofthe substance in d₆-DMSO. The ¹H and ¹³C NMR spectra were recorded on aBruker AMX-400 machine, the 2-D spectra were recorded using a BrukerDMX-600. The individual amino acids were assigned with the aid of theCOSY and the HMQC spectrum. With the aid of the COSY spectrum, theproton signals of the individual amino acids are assigned starting fromthe NH, or the α proton, by means of the H—H couplings. By using theHMQC spectrum, it is then possible to assign the carbon signals in asimilar manner. The sequence of the amino acids was determined with theaid of the NOESY and the HMBC spectrum. In the NOESY spectrum, thespatial distance of the NH of an amino acid to the α proton(s) of thenext amino acid is detected. In the HMBC spectrum, the α protons of twoamino acids which are linked to each other are correlated to the samecarbonyl carbon. The α protons also show two correlation peaks tocarbonyl carbons: one peak by two-bond coupling to the carbonyl carbonof its own amino acid and one peak by three-bond coupling to thecarbonyl carbon of the N-linked amino acid.

[0019] The following structure was determined:

[0020] The ¹H and ¹³C signals were assigned as shown in the two tablesbelow: TABLE 1 H atom δ/ppm Multiplicity rel. No. H H atom δ/ppmMultiplicity rel. No. H 58 10.73 D 1 39a 1.05 M 1 8 8.89 D 1 46b 0.94 M1 25 8.27 D 1 35 0.91 D 3 16 7.90 D 1 36 0.88 S 3 4 7.66 D 1 47 0.83 T 31 7.26 M 1 57 0.80 D 3 5 7.03 S 1 42 0.80 D 3 2 7.01 M 1 39b 0.78 M 1 36.92 M 1 45 0.79 D 3 22 5.09 D 1 53 0.77 D 3 12 5.03 D 1 49 0.75 D 3 145.02 D 1 51 0.74 T 3 7 5.00 M 1 43 0.73 D 3 18 4.92 D 1 38 0.71 D 3 294.88 D 1 54 0.67 D 3 9a 4.77 D 1 56 0.65 D 3 26a 4.59 D 1 40 0.64 T 3 272.81 S 3 32 0.60 D 3 30 2.76 S 3 33 0.21 D 3 11 2.72 S 3 24 4.48 T 1 132.71 S 3 15 4.46 T 1 19 2.59 S 3 20a 4.30 D 1 41 2.22 M 1 28 3.84 D 1 522.19 M 1 9b 3.66 D 1 55 2.11 M I 20b 3.46 M 1 48 2.06 M I 26b 3.30 D 137 2.02 M 1 6a 3.17 DD 1 44 1.98 M 1 23 3.01 S 3 31 1.95 M 1 17 2.97 S 334 1.90 M 1 21 2.93 S 3 50a 1.53 M 1 10 2.89 S 3 46a 1.18 M 1 6b 2.88 DDI 50b 1.07 M 1

[0021] TABLE 2 C atom δ/ppm Multiplicity rel. No. C C atom δ/ppmMultiplicity rel. No. C 39 23.1 T 1 34 29.9 D 1 11 28.9 Q 1 21 36.3 Q 147 11.0 Q 1 9 49.0 T 1 40 10.4 Q 1 7 49.3 D 1 49 14.1 Q 1 26 50.1 T 1 519.9 Q 1 20 50.7 T 1 56 17.0 Q 1 15 52.9 D 1 53 19,1 Q 1 24 53.8 D 1 3317.3 Q 1 22 55.3 D 1 54 17.5 Q 1 12 57.0 D 1 38 15.5 Q 1 18 57.5 D 1 4516.3 Q 1 14 57.7 D 1 32 18.4 Q 1 29 58.5 D 1 57 19.5 Q 1 28 63.9 D 1 4317.6 Q 1 XV 109.9 S 1 42 19.5 Q 1 1 110.9 D 1 19 29.1 Q 1 3 117.9 D 1 5025.0 Q 3 4 118.1 D 1 52 26.1 D 3 2 120.5 D 1 35 18.7 Q 3 5 123.5 D 1 3618.7 Q 3 XIV 127.5 S 1 41 26.1 D 3 XIII 135.9 S 1 48 34.1 D 1 IX 167.8 S1 37 34.1 D 1 VI 167.9 S 1 46 22.9 T 1 XII 167.9 S 1 13 29.0 Q 1 VII168.0 S 1 17 29.8 Q 1 VIII 168.4 S 1 31 27.0 D 1 II 168.4 S 1 27 36.0 Q1 IV 168.8 S 1 30 28.6 Q 1 V 168.8 S 1 23 30.5 Q 1 XI 169.6 S 1 10 36.0Q 1 1 170.9 S 1 44 32.0 D 1 X 172.0 S 1 6 28.0 T 1 III 172.3 S 1 55 25.7D 1

[0022] The structure of the novel compound according to the inventionwas determined by extensive analytical, in particular spectroscopic,studies. However, since errors in the interpretation of the analyticaldata of substances of complex structure cannot always be totallyexcluded, omphalotin is additionally described by some characteristicphysico-chemical data and spectra: a) Appearance: white powder b)Solubility readily soluble in methanol, acetone and 2- propanol, lessreadily soluble in ethyl acetate and hardly soluble in cyclohexane andt-butyl methyl ether c) Molecular weight: 1317 d) Empirical formulaC₆₉H₁₁₅N₁₃O₁₂ (elemental analysis): e) Spectra The data of the ¹H NMRspectra are shown in Tables 1 and 2 of the description.

[0023] According to the invention, the novel omphalotin is produced bythe fermentation of suitable strains of microorganisms from the class ofthe Basidiomycetes, in particular of the orders Omphalotus andLampteromyces, preferably Omphalotus olearius (synonym: Clitocybeilludens) and very particularly preferably of the Omphalotus oleariusstrains 83 039 (DSM 9737), 90 173 (DSM 9738), 91 050 (DSM 9739), 92 095(DSM 9740), 93 162 (DSM 9741) or 90 170 (DSM 9742) or by mutants orvariants thereof.

[0024] The fermentation process according to the invention is carriedout in a customary manner. It can be carried out with the aid of solid,semi-solid or liquid culture media. Preference is given to usingaqueous-liquid culture media.

[0025] The culture media are inoculated by customary methods, forexample using oblique tubes or flask cultures.

[0026] The cultivation is carried out under aerobic conditions and canbe carried out according to customary methods, for example by usingshaking cultures for example a shaking flask, by using air-agitatedcultures or submersion cultures. Preference is given to carrying out thecultivation using the aerobic submersion process in aerated fermenters,for example in customary submersion fermentation tanks. The cultivationcan be carried out continuously or batchwise. Preference is given tobatchwise operation.

[0027] The cultivation can be carried out in all culture media which areknown to be used for the cultivation of microorganisms of the class ofthe Basidiomycetes. The culture medium must contain one or moreassimilable carbon sources and nitrogen sources and mineral salts, itbeing possible for these products to be present in the form of definedindividual components, or else in the form of complex mixtures, asrepresented in particular by biological products of diverse origin.Suitable carbon sources are all customary carbon sources. Examplesinclude carbohydrates, in particular polysaccharides, such as starch ordextrins, disaccharides, such as maltose or cane sugar, monosaccharidessuch as glucose or xylose, sugar alcohols such as mannitol or glyceroland naturally occurring mixtures such as malt extract, molasses or wheypowder. Suitable nitrogen sources are all customary organic andinorganic nitrogen sources. Examples include proteins, proteinhydrolysates, amino acids such as glutamic acid, aspartic acid,arginine, lysine, ornithine or serine, nucleoside bases such as cytosineor uracil and soya bean meal, cotton seed meal, lentil meal, pea meal,soluble and insoluble plant proteins, maize steep liquor, yeast extract,peptones and meat extract and also ammonium salts and nitrates, forexample NH₄Cl, (NH₄)₂SO₄, NaNO₃ and KNO₃. The mineral salts which shouldbe present in the culture medium generate, for example, the followingions:

[0028] Mg⁺⁺, Na⁺, K⁺, Ca⁺⁺, NH₄ ⁺, Cl⁻, SO₄ ⁻⁻, PO₄ ⁻⁻⁻and NO₃ ⁻

[0029] and ions of the customary trace elements, such as Cu, Fe, Mn, Mo,Zn, Co, Ni. If the carbon or nitrogen sources or the water used do notcontain enough of these salts or trace elements it is useful tosupplement the culture medium appropriately. The composition of theculture media can vary within wide ranges. The kind and the compositionof the culture media will generally depend on which components are ineach case available at particularly low cost. In general, the culturesolutions contain preferably about 0.5 to 8%, in particular 0.6 to 6%,of carbon sources, preferably about 0.5 to 4%, in particular 0.5 to 2%,of nitrogen sources and preferably about 0.001 to 0.5%, in particular0.003 to 0.3%, of mineral salts.

[0030] When carrying out the process, it may be advantageous to use onlyrelatively low concentrations of the soluble culture solution componentsat the beginning of the cultivation, and to feed these components in theform of sterile, relatively concentrated solution to the culture brothby relatively frequent addition over the first 3 days of cultivation.

[0031] The pH of the growing cultures should preferably be maintained atbetween about 5 and about 10, in particular between 3.0 and 8.0. Anunacceptably large decrease in pH into the acid range can be avoided byaddition of an organic or inorganic base, preferably CaCO₃. As iscustomary in fermentation technology, it is also possible to carry outan automatic pH regulation where sterile organic or inorganic acid, forexample H₂SO₄, or sterile base, for example NaOH, is injected into theculture broth in intervals.

[0032] It is useful to make sure that the microorganisms are insufficient contact with oxygen and the nutrients. This can be ensured bycustomary methods such as shaking and stirring.

[0033] The cultivation temperature may be between about 15 and about 40°C., preferably between 20 and 35° C., particularly preferably, thetemperature is between about 22 and 27° C. The duration of thecultivation can vary within wide limits, depending, for example, on thecomposition of the culture medium and the cultivation temperature. Theparticular optimum conditions can easily be determined by any personskilled in the art of microbiology.

[0034] It has been found that the amount of the compound according tothe invention which becomes enriched in the cultivation broth generallyreaches its maximum after about 1 to 10, preferably about 4 to 7, daysafter the beginning of the cultivation. The desired end product offermentation can be determined with the aid ofthin-layer-chromatographical studies, with the aid of HPLC and UVabsorption spectra, in the plate diffusion test using a suitable fungusas test strain, or by the nematicidal or insecticidal activity.

[0035] As in all microbiological processes, extraneous infections of theculture media should be avoided. For this purpose, the usual precautionsare taken, such as sterilization of the culture media, of the culturevessels and of the air required for aeration. For the sterilization ofthe equipment, it is for example possible to use both steam and drysterilization, the temperatures preferably being at 100 to 140° C., inparticular at 120 to 130° C.

[0036] If foam is formed in undesirable quantities during thecultivation, the customary chemical defoamers, for example liquid fatsand oils, oil-water emulsions, paraffins, higher alcohols, such asoctadecanol, silicone oils, polyoxyethylene or polyoxypropylenecompounds (for example in amounts of up to about 1%) may be added. Foammay also be reduced or eliminated with the aid of the customarymechanical devices (which use, for example, centrifugal forces).

[0037] The compound according to the invention can be isolated bycustomary physico-chemical methods from the culture medium and from thebiomass. The isolation may be carried out for example by the customaryextraction processes, precipitation processes and/or chromatographicprocesses. The final purification of the isolated substance may also becarried out with the aid of the abovementioned methods. However, in manycases a final purification is not necessary, since minor impuritieswhich may be present do not adversely affect the activity of thecompound.

[0038] When carrying out the abovementioned isolation and purificationmethods, customary physico-chemical methods, for example measuring of acharacteristic band in the spectrum or of the R_(f) values,determination of the antimicrobial or of the nematicidal andinsecticidal activity, etc., may be employed to find the fractions inwhich the compound according to the invention is present in the highestconcentration or purity. These methods can also be employed to findsuitable microorganisms by routine processes.

[0039] The isolation and purification of the compound according to theinvention, for example in the case where a liquid aqueous culture mediumis used, can be carried out as follows:

[0040] After it is enriched in the supernatant of the culture, culturefiltrate and mycel are separated by customary methods (for examplecentrifugation).

[0041] The compound according to the invention can be isolated, and, ifappropriate, purified, from the culture filtrate, preferably from thebiomass, with the aid of customary extraction processes, precipitationprocesses and/or chromatographic processes. Chromatography can becarried out in the form of column chromatography.

[0042] Suitable for use as adsorbents are the customary inorganic ororganic adsorbents, such as, for example, alumina, silica gel, magnesiumsilicate, activated charcoal, cellulose, cellulose derivatives,synthetic resins such as polyamides, for example acetylated polyamide,dextran gels or modified dextran gels. Suitable for use as eluents areall the different solvents or solvent mixtures in which the compoundaccording to the invention is soluble. Preference is given to usingwater, ammonia solution, chloroform and methanol or mixtures thereof(for example mixtures of chloroform, methanol and aqueous NH₃ ormethanol and water).

[0043] For the isolation of the compound according to the invention,preference is given to using chromatographic processes, for examplenon-specific adsorption on sorbents such as silica gel, ion exchangechromatography or gel diffusion chromatography. These are methods whichare known from the purification of water-soluble charged naturalcompounds.

[0044] Furthermore, the countercurrent distribution (liquid-liquiddistribution) methods may also be employed advantageously.

[0045] The compound according to the invention can be obtained from itssolutions by customary methods, for example by the evaporation of thesolvent, freeze-drying, etc.

[0046] In a preferred embodiment of the invention, the biomass (themycel) is obtained by centrifugation of the fermentation material(culture broth and mycel) which is obtained by the aerobic cultivationof the strains at about 27° C.

[0047] The novel substance is preferably obtained by extraction of thebiomass. It can also be isolated from the culture filtrate by adsorptionon activated charcoal or on suitable resins. The most economical methodhas been proven to be the binding of the substance according to theinvention to unspecific adsorber resins based on polystyrene (forexample Amberlite XAD or Lewatit OC 1031). Desorption of the compoundsaccording to the invention is carried out fractionally, by mixtures ofwater and organic solvents, in particular water/methanol. The fractionswhich show activity in the test against Meloidogyne incognita areconcentrated under reduced pressure until the organic solvent has beencompletely removed and, if appropriate, lyophilized.

[0048] The lyophilized crude product is taken up in water and, afterinsoluble components have been separated off, purified further bycustomary chromatographic processes. Here, preference is given torenewed binding to absorber resins (for example Lewatit OC 1031), afurther purification of the active fractions by chromatography (forexample Sephadex LH 20). The novel substance is finally prepared in pureform by customary chromatographic methods, preferably by silica gelchromatography or preparative HPLC.

[0049] The compound according to the invention is suitable forcontrolling animal pests, in particular insects, arachnids andnematodes, encountered in agriculture, in forests, in the protection ofstored products and of materials, and in the hygiene sector. It ispreferably used as a crop protection agent. It is active againstnormally sensitive and resistant species and against all or some stagesof development. The abovementioned pests include:

[0050] From the order of the Isopoda, for example, Oniscus asellus,Armadillidium vulgare and Porcellio scaber.

[0051] From the order of the Diplopoda, for example, Blaniulusguttulatus.

[0052] From the order of the Chilopoda, for example, Geophiluscarpophagus and Scutigera spec.

[0053] From the order of the Symphyla, for example, Scutigerellaimmaculata.

[0054] From the order of the Thysanura, for example, Lepisma saccharina.

[0055] From the order of the Collembola, for example, Onychiurusarmatus.

[0056] From the order of the Orthoptera, for example, Blatta orientalis,Periplaneta americana, Leucophaea maderae, Blattella germanica, Achetadomesticus, Gryllotalpa spp., Locusta migratoria migratorioides,Melanoplus differentialis and Schistocerca gregana.

[0057] From the order of the Dermaptera, for example, Forficulaauricularia.

[0058] From the order of the Isoptera, for example, Reticulitermes spp.

[0059] From the order of the Anoplura, for example, Pediculus humanuscorporis, Haematopinus spp. and Linognathus spp.

[0060] From the order of the Mallophaga, for example, Trichodectes spp.and Damalinea spp.

[0061] From the order of the Thysanoptera, for example, Hercinothripsfemoralis and Thrips tabaci.

[0062] From the order of the Heteroptera, for example, Eurygaster spp.,Dysdercus intermedius, Piesma quadrata, Cimex lectularius, Rhodniusprolixus and Triatoma spp.

[0063] From the order of the Homoptera, for example, Aleurodesbrassicae, Bemisia tabaci, Trialeurodes vaporariorum, Aphis gossypii,Brevicoryne brassicae, Cryptomyzus ribis, Aphis fabae, Aphis pomi,Eriosoma lanigerum, Hyalopterus arundinis, Phylloxera vastatrix,Pemphigus spp., Macrosiphum avenae, Myzus spp., Phorodon humuli,Rhopalosiphum padi, Empoasca spp., Euscelis bilobatus, Nephotettixcincticeps, Lecanium corni, Saissetia oleae, Laodelphax striatellus,Nilaparvata lugens, Aonidiella aurantii, Aspidiotus hederae,Pseudococcus spp. and Psylla spp.

[0064] From the order of the Lepidoptera, for example, Pectinophoragossypiella, Bupalus piniarius, Cheimatobia brumata, Lithocolletisblancardella, Hyponomeuta padella, Plutella maculipennis, Malacosomaneustria, Euproctis chrysorrhoea, Lymantria spp., Bucculatrixthurberiella, Phyllocnistis citrella, Agrotis spp., Euxoa spp., Feltiaspp., Earias insulana, Heliothis spp., Spodoptera exigua, Mamestrabrassicae, Panolis flammea, Spodoptera litura, Spodoptera spp.,Trichoplusia ni, Carpocapsa pomonella, Pieris spp., Chilo spp., Pyraustanubilalis, Ephestia kuehniella, Galleria mellonella, Tineolabisselliella, Tinea pellionella, Hofmannophila pseudospretella, Cacoeciapodana, Capua reticulana, Choristoneura fumiferana, Clysia ambiguella,Homona magnanima and Tortrix viridana.

[0065] From the order of the Coleoptera, for example, Anobium punctatum,Rhizopertha dominica, Bruchidius obtectus, Acanthoscelides obtectus,Hylotrupes bajulus, Agelastica alni, Leptinotarsa decemlineata, Phaedoncochleariae, Diabrotica spp., Psylliodes chrysocephala, Epilachnavarivestis, Atomaria spp., Oryzaephilus surinamensis, Anthonomus spp.,Sitophilus spp., Otiorrhynchus sulcatus, Cosmopolites sordidus,Ceuthorrhynchus assimilis, Hypera postica, Dermestes spp., Trogodermaspp., Anthrenus spp., Attagenus spp., Lyctus spp., Meligethes aeneus,Ptinus spp., Niptus hololeucus, Gibbium psylloides, Tribolium spp.,Tenebrio molitor, Agriotes spp., Conoderus spp., Melolontha melolontha,Amphimallon solstitialis and Costelytra zealandica.

[0066] From the order of the Hymenoptera, for example, Diprion spp.,Hoplocampa spp., Lasius spp., Monomorium pharaonis and Vespa spp.

[0067] From the order of the Diptera, for example, Aedes spp., Anophelesspp., Culex spp., Drosophila melanogaster, Musca spp., Fannia spp.,Calliphora erythrocephala, Lucilia spp., Chrysomyia spp., Cuterebraspp., Gastrophilus spp., Hyppobosca spp., Stomoxys spp., Oestrus spp.,Hypoderma spp., Tabanus spp., Tannia spp., Bibio hortulanus, Oscinellafrit, Phorbia spp., Pegomyia hyoscyami, Ceratitis capitata, Dacus oleaeand Tipula paludosa.

[0068] From the order of the Siphonaptera, for example, Xenopsyllacheopis and Ceratophyllus spp.

[0069] From the order of the Arachnida, for example, Scorpio maurus andLatrodectus mactans.

[0070] From the order of the Acarina, for example, Acarus siro, Argasspp., Ornithodoros spp., Dermanyssus gallinae, Eriophyes ribis,Phyllocoptruta oleivora, Boophilus spp., Rhipicephalus spp., Amblyommaspp., Hyalomma spp., Ixodes spp., Psoroptes spp., Chorioptes spp.,Sarcoptes spp., Tarsonemus spp., Bryobia praetiosa, Panonychus spp. andTetranychus spp.

[0071] The phytoparasitic nematodes include, for example, Pratylenchusspp., Radopholus similis, Ditylenchus dipsaci, Tylenchulussemipenetrans, Heterodera spp., Globodera spp., Meloidogyne spp.,Aphelenchoides spp., Longidorus spp., Xiphinema spp. and Trichodorusspp.

[0072] Microbial pests which can be controlled with the aid of the novelcompound include in particular the phytopathogenic fungi:

[0073] Plasmodiophoromycetes, Oomycetes, Chytridiomycetes, Zygomycetes,Ascomycetes, Basidiomycetes and Deuteromycetes.

[0074] Phytopathogenic bacteria include in particular Pseudomonadaceae,Rhizobiaceae, Enterobacteriaceae, Corynebacteriaceae andStreptomycetaceae.

[0075] Some pathogens causing fungal and bacterial diseases which comeunder the generic names listed above are mentioned as examples, but notby way of limitation:

[0076] Xanthomonas species, such as, for example, Xanthomonas campestrispv. oryzae; Pseudomonas species, such as, for example, Pseudomonassyringae pv. lachrymans; Erwinia species, such as, for example, Erwiniaamylovora; Pythium species, such as, for example, Pythium ultimum;Phytophora species, such as, for example, Phytophthora infestans;

[0077] Pseudoperonospora species, such as, for example,Pseudoperonospora humuli or Pseudoperonospora cubense; Plasmoparaspecies, such as, for example, Plasmopara viticola; Peronospora species,such as, for example, Peronospora pisi or P. brassicae;

[0078] Erysiphe species, such as, for example, Erysiphe graminis;Sphaerotheca species, such as, for example, Sphaerotheca fuliginea;Podosphaera species, such as, for example, Podosphaera leucotricha;Venturia species, such as, for example, Venturia inaequalis; Pyrenophoraspecies, such as, for example, Pyrenophora teres or P. graminea (conidiaform: Drechslera, syn: Helminthosporium);

[0079] Cochliobolus species, such as, for example, Cochliobolus sativus(conidia form: Drechslera, syn: Helminthosporium); Uromyces species,such as, for example, Uromyces appendiculatus; Puccinia species, suchas, for example, Puccinia recondita; Tilletia species, such as, forexample, Tilletia caries; Ustilago species, such as, for example,Ustilago nuda or Ustilago avenae; Pellicularia species, such as, forexample, Pellicularia sasakii; Pyricularia species, such as, forexample, Pyricularia oryzae; Fusarium species, such as, for example,Fusarium culmorum;

[0080] Botrytis species, such as, for example, Botrytis cinerea;Septoria species, such as, for example, Septoria nodorum;

[0081] Leptosphaeria species, such as, for example, Leptosphaerianodorum; Cercospora species, such as, for example, Cercospora canescens;Alternaria species, such as, for example, Alternaria brassicae; andPseudocercosporella species, such as, for example, Pseudocercosporellaherpotrichoides. Furthermore, Helminthosporium carbonum may bementioned.

[0082] The compound of the formula (I) according to the invention inparticular has outstanding nematicidal activity, for example againstMeloidogyne incognita.

[0083] It has systemic action and can also be applied via the leaves.

[0084] The active compound can be converted to the customaryformulations, such as solutions, emulsions, wettable powders,suspensions, powders, dusting agents, pastes, soluble powders, granules,suspo-emulsion concentrates, natural and synthetic materials impregnatedwith active compound and very fine capsules in polymeric substances.

[0085] These formulations are produced in a known manner, for example bymixing the active compound with extenders, that is liquid solventsand/or solid carriers, if appropriate with the use of surfactants, thatis emulsifiers and/or dispersants and/or foam-formers.

[0086] If the extender used is water, it is also possible to use forexample organic solvents as auxiliary solvents. Suitable liquid solventsare essentially: aromatics, such as xylene, toluene oralkylnaphthalenes, chlorinated aromatics and chlorinated aliphatichydrocarbons, such as chlorobenzenes, chloroethylenes or methylenechloride, aliphatic hydrocarbons, for example mineral oil fractions,mineral and vegetable oils, alcohols, such as butanol or glycol andtheir ethers and esters, ketones, such as acetone, methyl ethyl ketone,methyl isobutyl ketone or cyclohexanone, strongly polar solvents, suchas dimethylformamide and dimethyl sulphoxide and water.

[0087] Suitable solid carriers are:

[0088] for example ammonium salts and ground natural minerals, such askaolins, clays, talc, chalk, quartz, attapulgite, montmorillonite ordiatomaceous earth, and ground synthetic minerals, such as finelydivided silica, alumina and silicates; suitable solid carriers forgranules are: for example crushed and fractionated natural rocks such ascalcite, marble, pumice, sepiolite and dolomite, and synthetic granulesof inorganic and organic meals, and granules of organic material such assawdust, coconut shells, maize cobs and tobacco stalks; suitableemulsifiers and/or foam-formers are: for example nonionic and anionicemulsifiers, such as polyoxyethylene fatty acid esters, polyoxyethylenefatty alcohol ethers, for example alkylaryl polyglycol ethers,alkylsulphonates, alkyl sulphates, arylsulphonates as well as proteinhydrolysates; suitable dispersants are: for example lignin-sulphitewaste liquors and methylcellulose.

[0089] Tackifiers such as carboxymethylcellulose and natural andsynthetic polymers in the form of powders, granules or latices, such asgum arabic, polyvinyl alcohol and polyvinyl acetate, and naturalphospholipids, such as cephalins and lecithins, and syntheticphospholipids, can be used in the formulations. Other additives can bemineral and vegetable oils.

[0090] It is possible to use colorants such as inorganic pigments, forexample iron oxide, titanium oxide and Prussian Blue, and organicdyestuffs, such as alizarin dyestuffs, azo dyestuffs and metalphthalocyanine dyestuffs, and trace nutrients such as salts of iron,manganese, boron, copper, cobalt, molybdenum and zinc.

[0091] The formulations in general contain between 0.1 and 95 percent byweight of active compound, preferably between 0.5 and 90%.

[0092] The active compound according to the invention can be present inits commercially available formulations and in the use forms, preparedfrom these formulations, as a mixture with other active compounds, suchas insecticides, attractants, sterilizing agents, bactericides,acaricides, nematicides, fungicides, growth-regulating substances orherbicides. The insecticides include, for example, phosphates,carbamates, carboxylates, chlorinated hydrocarbons, phenylureas andsubstances produced by microorganisms, inter alia.

[0093] Examples of particularly advantageous mixing components are thefollowing:

[0094] Fungicides:

[0095] 2-aminobutane; 2-anilino-4-methyl-6-cyclopropyl-pyrimidine;2′,6′-dibromo-2-methyl-4′-trifluoromethoxy-4′-trifluoro-methyl-1,3-thiazole-5-carboxanilide;2,6-dichloro-N-(4-trifluoromethylbenzyl)-benzamide;(E)-2-methoxyimino-N-methyl-2-(2-phenoxyphenyl)-acetamide;8-hydroxyquinoline sulphate; methyl(E)-2-{2-[6-(2-cyanophenoxy)-pyrimidin-4-yloxy]-phenyl}-3-methoxyacrylate;methyl (E)-methoxymino[alpha-(o-tolyloxy)-o-tolyl] acetate;2-phenylphenol (OPP), aldimorph, ampropylfos, anilazine, azaconazole,

[0096] benalaxyl, benodanil, benomyl, binapacryl, biphenyl, bitertanol,blasticidin-S, bromuconazole, bupirimate, buthiobate,

[0097] calcium polysulphide, captafol, captan, carbendazim, carboxin,quinomethionate, chloroneb, chloropicrin, chlorothalonil, chlozolinate,cufraneb, cymoxanil, cypro-conazole, cyprofuram,

[0098] dichlorophen, diclobutrazol, diclofluanid, diclomezine, dicloran,diethofencarb, difenoconazole, dimethirimol, dimethomorph, diniconazole,dinocap, diphenylamine, dipyrithione, ditalimfos, dithianon, dodine,drazoxolon,

[0099] edifenphos, epoxyconazole, ethirimol, etridiazole,

[0100] fenarimol, fenbuconazole, fenfuram, fenitropan, fenpiclonil,fenpropidin, fenpropimorph, fentin acetate, fentin hydroxide, ferbam,ferimzone, fluazinam, fludioxonil, fluoromide, fluquinconazole,flusilazole, flusulfamide, flutolanil, flutriafol, folpet,fosetyl-aluminium, fthalide, fuberidazole, furalaxyl, furmecyclox,

[0101] guazatine,

[0102] hexachlorobenzene, hexaconazole, hymexazol,

[0103] imazalil, imibenconazole, iminoctadine, iprobenfos (IBP),iprodione, isoprothiolane,

[0104] kasugamycin, copper preparations such as: copper hydroxide,copper naphthenate, copper oxychloride, copper sulphate, copper oxide,oxine-copper and Bordeaux mixture,

[0105] mancopper, mancozeb, maneb, mepanipyrim, mepronil, metalaxyl,metconazole, methasulfocarb, methfuroxam, metiram, metsulfovax,myclobutanil,

[0106] nickel dimethyldithiocarbamate, nitrothal-isopropyl, nuarimol,

[0107] ofurace, oxadixyl, oxamocarb, oxycarboxin,

[0108] pefurazoate, penconazole, pencycuron, phosdiphen, phthalide,pimaricin, piperalin, polycarbamate, polyoxin, probenazole, prochloraz,procymidone, propamocarb, propiconazole, propineb, pyrazophos,pyrifenox, pyrimethanil, pyroquilon,

[0109] quintozene (PCNB),

[0110] sulphur and sulphur preparations,

[0111] tebuconazole, tecloftalam, tecnazene, tetraconazole,thiabendazole, thicyofen, thiophanate-methyl, thiram, tolclofos-methyl,tolylfluanid, triadimefon, triadimenol, triazoxide, trichlamide,tricyclazole, tridemorph, triflumizole, triforine, triticonazole,

[0112] validamycin A, vinclozolin,

[0113] zineb, ziram.

[0114] Bactericides:

[0115] bronopol, dichlorophen, nitrapyrin, nickeldimethyldithiocarbamate, kasugamycin, octhilinone, furancarboxylic acid,oxytetracycline, probenazole, streptomycin, tecloftalam, copper sulphateand other copper preparations.

[0116] Insecticides/Acaricides/Nematicides:

[0117] abamectin, AC 303 630, acephate, acrinathrin, alanycarb,aldicarb, alphamethrin, amitraz, avermectin, AZ 60541, azadirachtin,azinphos A, azinphos M, azocyclotin,

[0118] Bacillus thuringiensis, bendiocarb, benfuracarb, bensultap,beta-cyfluthrin, bifenthrin, BPMC, brofenprox, bromophos A, bufencarb,buprofezin, butocarboxim, butylpyridaben,

[0119] cadusafos, carbaryl, carbofuran, carbophenothion, carbosulfan,cartap, CGA 157419, CGA 184699, chloethocarb, chlorethoxyfos,chlorfenvinphos, chlorfluazuron, chlormephos, chlorpyrifos, chlorpyrifosM, cis-resmethrin, clocythrin, clofentezine, cyanophos, cycloprothrin,cyfluthrin, cyhalothrin, cyhexatin, cypermethrin, cyromazine,

[0120] deltamethrin, demeton M, demeton S, demeton-S-methyl,diafenthiuron, diazinon, dichlofenthion, dichlorvos, dicliphos,dicrotophos, diethion, diflubenzuron, dimethoate, dimethylvinphos,dioxathion, disulfoton,

[0121] edifenphos, emamectin, esfenvalerate, ethiofencarb, ethion,ethofenprox, ethoprophos, etrimfos,

[0122] fenamiphos, fenazaquin, fenbutatin oxide, fenitrothion,fenobucarb, fenothiocarb, fenoxycarb, fenpropathrin, fenpyrad,fenpyroximate, fenthion, fenvalerate, fipronil, fluazinam,flucycloxuron, flucythrinate, flufenoxuron, flufenprox, fluvalinate,fonofos, formothion, fosthiazate, fubfenprox, furathiocarb,

[0123] HCH, heptenophos, hexaflumuron, hexythiazox,

[0124] imidacloprid, iprobenfos, isazofos, isofenphos, isoprocarb,isoxathion, ivermectin,

[0125] lambda-cyhalothrin, lufenuron,

[0126] malathion, mecarbam, mevinphos, mesulfenphos, metaldehyde,methacrifos, methamidophos, methidathion, methiocarb, methomyl,metolcarb, milbemectin, monocrotophos, moxidectin,

[0127] naled, NC 184, NI 25, nitenpyram,

[0128] omethoate, oxamyl, oxydemeton M, oxydeprofos,

[0129] parathion A, parathion M, permethrin, phenthoate, phorate,phosalone, phosmet, phosphamidon, phoxim, pirimicarb, pirimiphos M,pirimiphos A, profenofos, promecarb, propaphos, propoxur, prothiofos,prothoate, pymetrozine, pyrachlofos, pyridaphenthion, pyresmethrin,pyrethrum, pyridaben, pyrimidifen, pyriproxyfen,

[0130] quinalphos,

[0131] RH 5992,

[0132] salithion, sebufos, silafluofen, sulfotep, sulprofos,

[0133] tebufenozide, tebufenpyrad, tebupirimifos, teflubenzuron,tefluthrin, temephos, terbam, terbufos, tetrachlorvinphos, thiafenox,thiodicarb, thiofanox, thiomethon, thionazin, thuringiensin,tralomethrin, triarathene, triazophos, triazuron, trichlorfon,triflumuron, trimethacarb,

[0134] vamidothion, XMC, xylylcarb, YI 5301/5302, zetamethrin.

[0135] A mixture with other known active compounds, such as herbicides,or with fertilizers and growth-regulators is also possible.

[0136] The active compound according to the invention can furthermore bepresent in its commercially available formulations and in the use forms,prepared from these formulations, as a mixture with synergists.Synergists are compounds which increase the action of the activecompounds, without it being necessary for the synergist added to beactive itself.

[0137] The active compound content of the use forms prepared from thecommercially available formulations can vary within wide limits. Theactive compound concentration of the use forms can be from 0.0000001 to95% by weight of active compound, preferably between 0.0001 and 1% byweight.

[0138] The compound is employed in a customary manner appropriate forthe use forms. The active compound can be applied to above-ground partsof plants, or via the soil. Treatment of seed is also possible.

[0139] When used against hygiene and stored-product pests, the activecompound has an excellent residual action on wood and clay and a goodstability to alkali on limed substrates.

[0140] Having favourable toxicity to warm-blooded species, the activecompound is suitable for controlling pathogenic endoparasites whichoccur in humans and in particular in animal keeping and animal breeding,in productive animals, breeding animals, zoo animals, laboratoryanimals, animals for experimentation and pets. They are active againstall or individual stages of development of the pests and againstresistant and normally sensitive species. By controlling the pathogenicendoparasites the intention is to reduce disease, mortality andreductions in yield (for example in the production of meat, milk, wool,hides, eggs, honey, etc.), so that the use of the active compoundsenables more economical and simpler animal keeping. The pathogenicendoparasites include cestodes, trematodes, nematodes andAcantocephalea, in particular:

[0141] From the order of the Pseudophyllidea, e.g.: Diphyllobothriumspp., Spirometra spp., Schistocephalus spp., Ligula spp., Bothridiumspp., Diphlogonoporus spp.

[0142] From the order of the Cyclophyllidea, e.g.: Mesocestoides spp.,Anoplocephala spp., Paranoplocephala spp., Moniezia spp., Thysanosomasaspp., Thysaniezia spp., Avitellina spp., Stilesia spp., Cittotaeniaspp., Andyra spp., Bertiella spp., Taenia spp., Echinococcus spp.,Hydatigera spp., Davainea spp., Raillietina spp., Hymenolepis spp.,Echinolepis spp., Diorchis spp., Dipylidium spp., Joyeuxiella spp.,Diplopylidium spp.

[0143] From the subclass of the Monogenea, e.g.: Gyrodactylus spp.,Dactylogyrus spp., Polystoma spp.

[0144] From the subclass of the Digenea, e.g.: Diplostomum spp.,Posthodiplostomum spp., Schistomsoma spp., Trichobilharzia spp.,Ornithobilharzia spp., Austrobilharzia spp., Gigantobilharzia spp.,Leucochloridium spp., Brachylaima spp., Echinostoma spp.,Echinoparyphium spp., Echinochasmus spp., Hypoderaeum spp., Fasciolaspp., Fasciolides spp., Fasciolopsis spp., Cyclocoelum spp.,Typhlocoelum spp., Paramphistomum spp., Calicophoron spp., Cotylophoronspp., Gigantocotyle spp., Fischoederius spp., Gastrothylacus spp.,Notocotylus spp., Catatropis spp., Plagiochis spp., Prosthogonimus spp.,Dicrocoelium spp., Eurytrema spp., Troglotrema spp., Paragomimus spp.,Collyriclum spp., Nanophyetus spp., Opisthorchis spp., Clonorchis spp.,Metorchis spp., Heterophyes spp., Metagonismus spp.

[0145] From the order of the Enoplida e.g.: Trichuris spp., Capillariaspp., Trichomosoides spp., Trichinella spp.

[0146] From the order of the Rhabditia e.g.: Micronema spp.,Strongyloides spp.

[0147] From the order of the Strongylida e.g.: Stronylus spp.,Triodontophorus spp., Oesophagodontus spp., Trichonema spp.,Gyalocephalus spp., Cylindropharynx spp., Poteriostomum spp.,Cyclococercus spp., Cylicostephanus spp., Oesophagostomum spp.,Chabertia spp., Stephanurus spp., Ancylostoma spp., Uncinaria spp.,Bunostomum spp.,

[0148] Globocephalus spp., Syngamus spp., Cyathostoma spp.,Metastrongylus spp., Dictyocaulus spp., Muellerius spp., Protostrongylusspp., Neostrongylus spp., Cystocaulus spp., Pneumostrongylus spp.,Spicocaulus spp., Elaphostrongylus spp., Parelaphostrongylus spp.,Crenosoma spp., Paracrenosoma spp., Angiostrongylus spp.,Aelurostrongylus spp., Filaroides spp., Parafilaroides spp.,Trichostrongylus spp., Haemonchus spp., Ostertagia spp., Marshallagiaspp., Cooperia spp., Nematodirus spp., Hyostrongylus spp., Obeliscoidesspp., Amidostomum spp., Ollulanus spp.

[0149] From the order of the Oxyurida e.g.: Oxyuris spp., Enterobiusspp., Passalurus spp., Syphacia spp., Aspiculuris spp., Heterakis spp.

[0150] From the order of the Ascaridia e.g.: Ascaris spp., Toxascarisspp., Toxocara spp., Parascaris spp., Anisakis spp., Ascaridia spp.

[0151] From the order of the Spirurida e.g.: Gnathostoma spp.,Physaloptera spp., Thelazia spp., Gongylonema spp., Habronema spp.,Parabronema spp., Draschia spp., Dracunculus spp.

[0152] From the order of the Filariida e.g.: Stephanofilaria spp.,Parafilaria spp., Setaria spp., Loa spp., Dirofilaria spp., Litomosoidesspp., Brugia spp., Wuchereria spp., Onchocerca spp.

[0153] From the order of Gigantorhynchida e.g.: Filicollis spp.,Moniliformis spp., Macracanthorhynchus spp., Prosthenorchis spp.

[0154] The productive and breeding animals include mammals such ascattle, horses, sheep, pigs, goats, camels, water buffalo, donkeys,rabbits, fallow deer and reindeer, fur-bearing animals such as mink,chinchilla and raccoon, birds such as chicken, geese, turkeys and ducks,fresh- and salt-water fish such as trout, carp and eels, reptiles,insects such as honey-bee and silkworm.

[0155] Laboratory and experimental animals include mice, rats,guinea-pigs, golden hamsters, dogs and cats.

[0156] The pets include dogs and cats.

[0157] Administration can be carried out both prophylactically andtherapeutically.

[0158] Administration of the active compound is carried out directly orin the form of suitable preparations, enterally, parenterally, dermally,nasally, by treating the habitat or with the aid of activecompound-containing shaped articles such as, for example, stripes,plates, bands, collars, ear-tags, straps for limbs, marking devices.

[0159] Enteral administration of the active compound is carried out, forexample, orally in the form of powders, tablets, capsules, pastes,drinks, granules, orally administrable solutions, suspensions andemulsions, boluses, medicated feed or drinking water. Dermaladministration is carried out, for example, in the form of dipping,spraying or pouring-on and spotting-on. Parenteral administration iscarried out, for example, in the form of injections (intramuscular,subcutaneous, intravenous, intraperitoneal), or by implants.

[0160] Suitable preparations are:

[0161] solutions such as solutions for injection, oral solutions,concentrates for oral administration after dilution, solutions for useon the skin or in body cavities, pouring-on formulations, gels;

[0162] emulsions and suspensions for oral or dermal administration andfor injection; semi-solid preparations;

[0163] formulations in which the active compound is processed in anointment base or in an oil-in-water or water-in-oil emulsion base;

[0164] solid preparations such as powders, premixes or concentrates,granules, pellets, tablets, boluses, capsules; aerosols and inhalants,active compound-containing shaped articles.

[0165] Solutions for injection are administered intravenously,intramuscularly, and subcutanueously.

[0166] Solutions for injection are prepared by dissolving the activecompound in a suitable solvent and adding, if appropriate, additivessuch as solubilizers, acids, bases, buffer salts, antioxidants,preservatives. The solutions are filtered and filled under sterileconditions.

[0167] Solvents which may be mentioned are: physiologically tolerablesolvents such as water, alcohols such as ethanol, butanol, benzylalcohol, glycerol, propylene glycol, polyethylene glycols,N-methyl-pyrrolidone, and mixtures thereof.

[0168] The active compound can optionally also be dissolved inphysiologically tolerable vegetable or synthetic oils which are suitablefor injection.

[0169] Solubilizers which may be mentioned are: solvents which promotethe dissolution of the active compound in the main solvent or preventits precipitation. Examples are polyvinylpyrrolidone, polyoxyethylatedcastor oil, polyoxyethylated sorbitan ester.

[0170] Preservatives are: benzyl alcohol, trichlorobutanol,p-hydroxybenzoic acid esters, n-butanol.

[0171] Oral solutions are administered directly. Concentrates areadministered orally after prior dilution to the use concentration. Oralsolutions and concentrates are prepared as described above for thesolutions for injection, sterile procedures not being necessary.

[0172] Solutions for use on the skin are trickled on, spread on, rubbedin, sprinkled on or sprayed on. These solutions are prepared asdescribed above for the solutions for injection.

[0173] It may be advantageous to add thickeners during preparation.Thickeners are: inorganic thickeners such as bentonites, colloidalsilicic acid, aluminium monostearate, organic thickeners such ascellulose derivatives, polyvinyl alcohols and their copolymers,acrylates and methacrylates.

[0174] Gels are applied to or spread on the skin or introduced into bodycavities. Gels are prepared by treating solutions which have beenprepared as described in the case of the injection solutions withsufficient thickener that a clear material having an ointment-likeconsistency results. The thickeners employed are the thickeners givenabove.

[0175] Pour-on formulations are poured or sprayed onto limited areas ofthe skin, the active compound penetrating the skin and actingsystemically.

[0176] Pour-on formulations are prepared by dissolving, suspending oremulsifying the active compound in suitable skin-compatible solvents orsolvent mixtures. If appropriate, other auxiliaries such as colorants,bioabsorption-promoting substances, antioxidants, light stabilizers,adhesives are added.

[0177] Solvents which may be mentioned are: water, alkanols, glycols,polyethylene glycols, polypropylene glycols, glycerol, aromatic alcoholssuch as benzyl alcohol, phenylethanol, phenoxyethanol, esters such asethyl acetate, butyl acetate, benzyl benzoate, ethers such as alkyleneglycol alkyl ethers such as dipropylene glycol monomethyl ether,diethylene glycol mono-butyl ether, ketones such as acetone, methylethyl ketone, aromatic and/or aliphatic hydrocarbons, vegetable orsynthetic oils, DMF, dimethylacetamide, N-methylpyrrolidone and2,2-dimethyl-4-oxy-methylene-1,3-dioxolane.

[0178] Colorants are all colorants permitted for use on animals andwhich can be dissolved or suspended.

[0179] Absorption-promoting substances are, for example, DMSO, spreadingoils such as isopropyl myristate, dipropylene glycol pelargonate,silicone oils, fatty acid esters, triglycerides, fatty alcohols.

[0180] Antioxidants are sulfites or metabisulfites such as potassiummetabisulfite, ascorbic acid, butylhydroxytoluene, butylhydroxyanisole,tocopherol.

[0181] Light stabilizers are, for example, novantisolic acid.

[0182] Adhesives are, for example, cellulose derivatives, starchderivatives, polyacrylates, natural polymers such as alginates, gelatin.

[0183] Emulsions can be administered orally, dermally or as injections.

[0184] Emulsions are either of the water-in-oil type or of theoil-in-water type.

[0185] They are prepared by dissolving the active compound either in thehydrophobic or in the hydrophilic phase and homogenizing this with thesolvent of the other phase with the aid of suitable emulsifiers and, ifappropriate, other auxiliaries such as colorants,bioabsorption-promoting substances, preservatives, antioxidants, lightstabilizers, viscosity-enhancing substances.

[0186] Hydrophobic phases (oils) which may be mentioned are: liquidparaffins, silicone oils, natural vegetable oils such as sesame oil,almond oil, castor oil, synthetic triglycerides such as caprylic/capricbiglyceride, triglyceride mixture with vegetable fatty acids of thechain length C₈₋₁₂ or other specially selected natural fatty acids,partial glyceride mixtures of saturated or unsaturated fatty acidspossibly also containing hydroxyl groups, mono- and diglycerides of theC₈/C₁₀ fatty acids.

[0187] Fatty acid esters such as ethyl stearate, di-n-butyryl adipate,hexyl laurate, dipropylene glycol perlargonate, esters of a branchedfatty acid of medium chain length with saturated fatty alcohols of chainlength C₁₆-C₁₈, isopropyl myristate, isopropyl palmitate,caprylic/capric acid esters of saturated fatty alcohols of chain lengthC₁₂-C₁₈, isopropyl stearate, oleyl oleate, decyl oleate, ethyl oleate,ethyl lactate, waxy fatty acid esters such as synthetic duck coccygealgland fat, dibutyl phthalate, diisopropyl adipate, ester mixturesrelated to the latter, inter alia.

[0188] Fatty alcohols such as isotridecyl alcohol, 2-octyldodecanol,cetylstearyl alcohol, oleyl alcohol.

[0189] Fatty acids such as oleic acid and its mixtures.

[0190] Hydrophilic phases which may be mentioned are: water, alcoholssuch as propylene glycol, glycerol, sorbitol and its mixtures.

[0191] Emulsifiers which may be mentioned are: non-ionic surfactants,e.g. polyethoxylated castor oil, polyethoxylated sorbitan monooleate,sorbitan monostearate, glycerol monostearate, polyoxyethyl stearate,alkylphenol polyglycol ether;

[0192] ampholytic surfactants such as di-Na N-lauryl-β-iminodipropionateor lecithin;

[0193] anionic surfactants, such as Na lauryl sulphate, fatty alcoholether sulphates, mono/dialkyl polyglycol ether orthophosphoric acidester monoethanolamine salt.

[0194] Further auxiliaries which may be mentioned are: substances whichenhance the viscosity and stabilize the emulsion, such ascarboxymethylcellulose, methylcellulose and other cellulose and starchderivatives, polyacrylates, alginates, gelatin, gum arabic,polyvinylpyrrolidone, polyvinyl alcohol, copolymers of methyl vinylether and maleic anhydride, polyethylene glycols, waxes, colloidalsilicic acid or mixtures of the substances mentioned.

[0195] Suspensions can be administered orally, dermally or as injection.They are prepared by suspending the active compound in a suspendingagent, if appropriate with addition of other auxiliaries such as wettingagents, colorants, bioabsorption-promoting substances, preservatives,antioxidants, light stabilizers.

[0196] Liquid excipients which may be mentioned are all homogeneoussolvents and solvent mixtures.

[0197] Wetting agents (dispersants) which may be mentioned are thesurfactants given above.

[0198] Other auxiliaries which may be mentioned are those given above.

[0199] Semi-solid preparations can be administered orally or dermally.They differ from the suspensions and emulsions described above only bytheir higher viscosity.

[0200] For the production of solid preparations, the active compound ismixed with suitable excipients, if appropriate with addition ofauxiliaries, and brought into the desired form.

[0201] Excipients which may be mentioned are all physiologicallytolerable solid inert substances. Those used are inorganic and organicsubstances. Inorganic substances are, for example, sodium chloride,carbonates such as calcium carbonate, hydrogencarbonates, aluminiumoxides, silicic acids, argillaceous earths, precipitated or colloidalsilica, phosphates.

[0202] Organic substances are, for example, sugar, cellulose, foodstuffsand feeds such as milk powder, animal meal, grain meals and shreds,starches.

[0203] Auxiliaries are preservatives, antioxidants, colorants which havealready been mentioned above.

[0204] Other suitable auxiliaries are lubricants and glidants such asmagnesium stearate, stearic acid, talc, bentonites,disintegration-promoting substances such as starch or crosslinkedpolyvinylpyrrolidone, binders such as starch, gelatin or linearpolyvinylpyrrolidone, and dry binders such as microcrystallinecellulose.

[0205] The active compounds can also be present in the preparations as amixture with synergists or with other active compounds which act againstpathogenic endoparasites. Such active compounds are, for example,L-2,3,5,6-tetrahydro-6-phenylimidazothiazole, benzimidazole carbamates,praziquantel, pyrantel, epsiprantel.

[0206] Ready-to-use preparations contain the active compound inconcentrations of 10 ppm-20 percent by weight, preferably of 0.1-10percent by weight.

[0207] Preparations which are diluted before use contain the activecompound in concentrations of 0.5-90% by weight, preferably of 5-50% byweight.

[0208] In general, it has proven to be advantageous to administeramounts of about 1 to about 100 mg of active compound per kg of bodyweight per day to obtain effective results.

[0209] The preparation of the novel omphalotin and its biologicalproperties are illustrated by the following examples.

[0210] In the examples (as in the rest of the description) percentagesrefer to percentages by weight and specifications of solvent mixturesrefer to parts by volume, unless stated otherwise.

[0211] The test methods given in the examples are also particularlysuitable to determine the omphalotin-containing fractions in theproduction of omphalotin. They can also be employed to estimate theparticular omphalotin content. Example 1 (Preparation of omphalotin byfermentation of Omphalotus olearius No. 90 170 = DSM 9742) 1.1Preparation of solid media agar plates for the cultivation of Omphalotusolearius 1.2 Cultivation of Omphalotus olearius on solid media agarplates 1.3 Preparation of the culture medium for the precultivation ofOmphalotus olearius 1.4 Inoculation and cultivation of Omphalotusolearius in the preculture medium 1.5 Preparation of the culture mediumfor the fermentation of Omphalotus olearius in the prefermenter 1.6Inoculation and cultivation of Omphalotus olearius in the prefermenter1.7 Preparation of the culture medium for the fermentation of Omphalotusolearius in the main fermenter 1.8 Inoculation and cultivation ofOmphalotus olearius in the main fermenter 1.9 Harvest of the biomassafter the fermentation

[0212] 1.1 Preparation of Solid Media Agar Plates for the Cultivation ofOmphalotus olearius

[0213] For the preparation of solid media agar plates

[0214] 4 g/l of glucose

[0215] 4 g/l of yeast extract (Merck No. 3753, 64271 Darmstadt, Germany)

[0216] 10 g/l of Löflund's barley malt extract (Dr. Fränkle & Max Eck,70736 Fellbach, Germany)

[0217] 20 g/l of agar agar (Merck No. 1615, 64271 Darmstadt, Germany)

[0218] are weighed in, dissolved in demineralized water and adjusted topH 5.5 by addition of acid (c(HCl)=2 mol/l). A 2 l Erlenmeyer flask ischarged with 1 l of the abovementioned solution, sealed with a cottonplug and sterilized in an autoclave (MMM, type LSS 666-1) at atemperature of 121° C. for 30 minutes.

[0219] With continuous stirring, the solution is cooled to a temperatureof about 50 to 60° C. 20 ml of the solution are transferred into 90 mmPetri dishes under sterile conditions. After the solution has cooled,the filled Petri dishes can be stirred at room temperature for a maximumof 8 weeks prior to an inoculation.

[0220] 1.2 Cultivation of Omphalotus olearius on Solid Media Agar Plates

[0221] The agar of a solid media agar plate which is densely colonizedby Omphalotus olearius (in the present example No. 90 170) is dividedunder sterile conditions into pieces of a size of 1×1 cm with the aid ofa scalpel. In each case one agar mycel piece is transferred with a pairof tweezers onto a solid media agar plate as described under 1.1 (mycelfacing upwards) and incubated in an incubation cabinet (Heraeus, type BK5060 E) at 27° C. in the dark for a maximum of 3 weeks. After 3 weekshave expired, the colonized plates can be used as inoculation plates forthe fermentation, or they can be used once more for the inoculation ofsolid media agar plates.

[0222] 1.3 Preparation of the Culture Medium for the Precultivation ofOmphalotus olearius

[0223] 4 g/l of glucose

[0224] 4 g/l of yeast extract (Merck No. 3753, 64271 Darmstadt, Germany)

[0225] 10 g/l of Löflund's barley malt extract (Dr. Fränkle & Max Eck,70736 Fellbach, Germany)

[0226] are weighed in, dissolved in demineralized water and adjusted topH 5.5 by addition of acid (c(HCl)=2 mol/l). A 1 l Erlenmeyer flask ischarged with 300 ml of the abovementioned solution, sealed with a cottonplug and autoclaved at 121° C. for 30 minutes. After cooling, the filledErlenmeyer flasks are stored at room temperature for a maximum of 8weeks prior to an inoculation.

[0227] 1.4 Inoculation and Cultivation of Omphalotus olearius in thePreculture Medium

[0228] In each case 300 ml of the culture medium described under 1.3 areinoculated with half of a densely populated 2- to 3-week-old solid mediaplate. To this end, 2- to 3-week-old solid media plates are dividedunder sterile conditions using a scalpel and transferred into a Waringmixer attachment (Waring Blender, type 32BL79) by means of a pair oftweezers. The solid media plates are mixed with 50 ml of steriledistilled water per solid media plate and homogenized for 30 seconds (15seconds on low setting, 15 seconds on high setting). The total volume ofthe homogenized liquid is distributed evenly, using a 10 ml pipette(Becton Dickinson and Company, type Falcon), over the Erlenmeyer flaskwhich had been prepared for inoculation. The incubation of thepreculture flasks is carried out in a shaking cabinet (Braun Melsungentype BS 4) at a temperature of 27° C. and a shaking speed of 120 rpm for5 days.

[0229] 1.5 Preparation of the Culture Medium for the Fermentation ofOmphalotus olearius in the Prefermenter

[0230] A 42 l prefermenter (Braun Melsungen type Biostat P) is filledwith 30 l of demineralized water, and

[0231] 4 g/l of glucose

[0232] 4 g/l of yeast extract (Merck No. 3753, 64271 Darmstadt, Germany)

[0233] 10 g/l of Löflund's barley malt extract (Dr. Fränkle & Max Eck,70736 Fellbach, Germany)

[0234] 0.25 g/l of anti-foam agent (Bayer, Baysilone E, 51371Leverkusen, Germany)

[0235] are added. After filling, the fermenter is closed and the contentis sterilized at a temperature of 121° C. for 30 minutes. After coolingto an operating temperature of 27° C., an overpressure of 0.3 bar isapplied and the aeration rate is set to 0.25 vvm. After the oxygensaturation has reached the maximum, the pO₂ meter is calibrated to aninitial value of 99%.

[0236] The pH of the medium is adjusted to 5.5 with the aid of acid(c(HCl)=2 mol/l).

[0237] 1.6 Inoculation and Cultivation of Omphalotus olearius in thePrefermenter

[0238] The prefermenter is inoculated with 600 ml of the precultureprepared under 1.4. To this end, two 1 l Erlenmeyer flasks, eachcontaining 300 ml of preculture, are transferred under sterileconditions into a 2 l glass bottle (Schott) (the bottle is connected toa Braun Melsungen inoculating apparatus via a silicone tube).

[0239] By means of this inoculating apparatus, the prefermenter isinoculated with the preculture under sterile conditions through amembrane in the lid.

[0240] Fermentation conditions: Temperature 27° C. Stiffer speed 200 rpmAeration rate 0.25 vvm Pressure 0.3 bar pH not controlled Antifoam asrequired Incubation time about 120 h, or at the most up to the pointwhere the glucose concentration has decreased to c(glucose) ≦ 1 g/l.

[0241] 1.7 Preparation of the Culture Medium for the Fermentation ofOmphalotus olearius in the Main Fermenter

[0242] A 450 l fermenter (Braun Melsungen type Biostat 450 D) is filledwith 320 l of demineralized water, and

[0243] 4 g/l of glucose

[0244] 4 g/l of Fermtech yeast extract (Merck No. 11926, 64271Darmstadt, Germany)

[0245] 10 g/l of Löflund's barley malt extract (Dr. Fränkle & Max Eck,70736 Fellbach, Germany)

[0246] 0.25 g/l of anti-foam agent (Bayer, Baysilone E, 51371Leverkusen, Germany)

[0247] are added, based on the operating volume of 350 l. After filling,the fermenter is closed and the content is sterilized at a temperatureof 121° C. for 30 minutes. After cooling to an operating temperature of27° C., an overpressure of 0.3 bar is applied and the aeration rate isset to 0.25 vvm. After the oxygen saturation has reached the maximum,the pO₂ meter is calibrated to an initial value of 99%.

[0248] The pH of the medium is adjusted to 5.5 with the aid of acid(c(HCI)=2 mol/l).

[0249] 1.8 Inoculation and Cultivation of Omphalotus olearius in theMain Fermenter

[0250] After the glucose concentration c(glucose) in the culturefiltrate of the prefermenter (see 1.6) has dropped to below≦1 g/l, themain fermenter is inoculated with the content of the prefermenter viasterile transfer tubing. The glucose concentration is determined using aBeckmann glucose analyser.

[0251] Fermentation conditions: Temperature 27° C. Stiffer speed 150 rpmAeration rate 0.25 vvm Pressure 0.3 bar pH not controlled Antifoam asrequired Incubation time 72 h to 96 h

[0252] 1.9 Harvest of the Biomass After the Fermentation

[0253] At a glucose concentration of c(glucose)≦ 1 g/l, the fermentationin the 450 l fermenter is terminated and the biomass is harvested. Thecontent of the main fermenter is separated from the culture mediumthrough gauze (Holthaus Medical, Verbandmull, Remscheid 11, Germany).The biomass filtercake is washed repeatedly with demineralized water andsubsequently centrifuged in a tumble drier (Miele, type MZ 5942,Germany) at 4000 rpm for 3 minutes. 3 to 6 kg of moist biomass areharvested. The Omphalotus olearius biomass obtained is frozen at atemperature of −34° C. and stored until further work-up.

[0254] The fermentation of the other Omphalotus strains can be carriedout similarly to the example above.

EXAMPLE 2 Isolation and Purification of Omphalotin

[0255] 2.1 Preparation of the Crude Extract of the Active Compound fromOmphalotus olearius and Enrichment by Liquid-Liquid Distribution(Countercurrent Distribution)

[0256] About 7 kg of mycel pellets (biomass) from the mycel obtained byExample 1 were homogenized a little at a time and digested twice with 20l of acetone each time. The mixture was subsequently concentrated underreduced pressure using a rotary evaporator. 3 l of water and 3 l ofethyl acetate were added to the residue, which was then stirred. Theupper phase was separated off and the solvent was distilled off underreduced pressure using a rotary evaporator, the lower phase wasdiscarded. The distillation residue was taken up twice in 2 l ofacetonitrile and 0.5 l of n-heptane each time. The mixtures were shakenand allowed to stand, and the lower phases that had formed wereevaporated at about 40° C. The residue that remained was used for thecountercurrent distribution, which was carried out as described below.

[0257] Parameters for the countercurrent distribution: Apparatus: 25 mlapparatus (Labortec, Bubendorf, Switzerland) Number of 200 distributionelements: Distribution ethyl acetate/n-heptane/DMF(dimethylformamide)/water system: (2.5/7.5/5/5 v/v) Phase ratio: 1:1Separation n = 198 stages: Charge: 945 mg in two elements Work-up: Afterthe end of the distribution cycle (n = 198), the content of every 10thelement was removed, the solvents were distilled off under reducedpressure using a rotary evaporator, and the residue was weighed and thentaken up in 10 ml of methanol and examined by analytical HPLC (seeExample 2.4). The omphalotin was found in elements E 48-87. The contentof the elements was removed and combined. 2 l of water were added, themixture was shaken and the upper phase was separated off. The lowerphase was extracted 3 times with 1 l of ethyl acetate p.a. each time.The combined upper phases were then extracted 4 times with 0.5 l ofwater each time, and the solvent was distilled off under reducedpressure using a rotary evaporator. The yield of the concentrated crudeextract that remained was 135 mg. Further purification was carried outas in Example 2.3.

[0258] 2.2. Preparation of the Crude Extract of the Active Compound fromOmphalotus olearius and Enrichment of the Active Compound by thePrinciple of Gel Filtration

[0259] 400 g of lyophilized mycel were extracted with stirring with 10 lof methanol for 2 hours. The solvent was removed using a rotaryevaporator, yielding 35 g of crude extract. This crude extract wasdivided into portions of 10 g.

[0260] 10 g of crude extract were dissolved in 5 ml of methanol andchromatographed over a separation column (height: 62 cm, diameter: 5 cm)filled with Sephadex LH-20 (Pharmacia, Upsalla, Sweden), using methanolas mobile phase. The flow rate was adjusted so that 5.5 ml/min of eluatewere obtained. The eluate was collected in 100 ml fractions. Thefractions containing omphalotin were combined and evaporated using arotary evaporator. The original 10 g yielded 2 g of enriched extract.The 2 g of enriched extract were divided into portions of 200 mg.

[0261] 200 mg of the abovementioned enriched extract were dissolved in 1ml of methanol and applied to a second separation column (height 71.5cm, diameter 2.5 cm, Sephadex LH-20). Chromatography was then carriedout using a flow rate of 3 ml/min. The eluate was collected in 10 mlfractions. These fractions were likewise examined for omphalotin usinganalytical HPLC (see 2.4). Alternatively, the omphalotin could also bedetected using a bioassay, for example as in Example B. Theomphalotin-containing fractions were combined and evaporated using arotary evaporator. The original 200 mg yielded 60 mg of further enrichedextract. Further purification was carried out as in 2.3.

[0262] 2.3 Final Separation by Preparative HPLC of the Enriched ActiveCompound Fractions Obtained from the Liquid-Liquid Distribution and theChromatographic Separations Apparatus: Jasco PU-980 fitted with highpressure pump LG-980-02 gradient mixer and multiwavelength detectorMD-910 (Gross-Umstadt, Germany) Column: Hibar RT Lichrospher RP 18 WP300 ® (particle diameter 12 μm), Dimension: 250 mm column length * 25 mminternal diameter Eluent: A = water, B = acetonitrile Flow rate: 5ml/min Detection: UV, λ = 210 nm

[0263] Amount injected: 100 mg dissolved in 1 ml of acetonitrile

[0264] Gradients (all values in % by volume): Gradient 1: Gradient 2:  0min 100% A  0% B  0 min 100% A  0% B  70 min  70% A  30% B  40 min  70%A  30% B 100 min  40% A  60% B 100 min  40% A  60% B 160 min  0% A 100%A 160 min  0% A 100% B 200 min  0% A 100% A 200 min  0% A 100% B 220 min100% A  0% A 220 min 100% A  0% B

[0265] The samples from work-up 2.2 or 2.3 were initiallychromatographed using gradient 1. To this end, 100 mg of the testsubstance were applied to the column in 1 ml of acetonitrile andchromatographed. The substantially pure active compound (omphalotin)eluted between 100 and 160 min at 30% of A. This fraction (= furtherpurified product) was subsequently rechromatographed using gradient 2.The pure active compound was in the fraction which appeared between 100and 160 min at 30% of B. The fraction containing pure active compoundwas freed from the solvent by distillation under reduced pressure usinga rotary evaporator. Starting from 400 g of mycel, 11.5 mg of the pureactive compound were obtained after the abovementioned steps had beencarried out repeatedly.

[0266] 2.4 Analytical High Pressure Liquid Chromatography (HPLC)Apparatus: HPLC 1090 having a diode array detector, Hewlett Packard(Waldbronn, Germany) Column: Lichrocart, Lichrospher RP 18 WP 300 ®(particle diameter 5 μm) Column dimension: 250*4 mm Eluent: A = water, B= acetonitrile Flow rate: 1 ml/mm Temperature: 40° C. Detection: UV, λ =210 nm Amount injected: 10 μl of a 0.1% strength solution (percent byweight) of the sample in acetonitrile

[0267] Gradient: Gradient  0 min  0% B 100% A 15 min  60% B  30% A 30min 100% B  0% A 35 min 100% B  0% A 40 min  0% B 100% A

[0268] Retention time for omphalotin under these conditions 20.8 min.

[0269] The fractions to be analysed were evaporated using a rotaryevaporator, and then taken up in acetonitrile and injected onto thecolumn as a 0.1% solution. The retention time for omphalotin was 20.8min. Detection was by the UV spectrum which is automatically determinedby the apparatus for the eluate.

EXAMPLE A Effect of Omphalotin on Heterodera schachtii

[0270] Test nematode: Heterodera schachtii

[0271] Test method:

[0272] The test substance is dissolved in methanol and pipetted into thewells of tissue culture plates (24 well tray). The methanol is thenallowed to evaporate completely. Each well is filled with 0.8 ml ofphysiological sodium chloride solution. Subsequently, 0.2 ml of anematode suspension (500 nematodes/ml) are pipetted into each well.

[0273] After 24 hours of incubation at room temperature and gentleshaking of the plates, the number of live and dead nematodes is counted.

[0274] The efficacy of the test substance is 100% if all test nematodeshave been killed, and it is 0% if the number of nematodes that are stillalive is the same as in the control.

[0275] The following efficacies were found: Omphalotin concentration(ppm = mg/l) Efficacy (in % Abbott) 10 ppm 60  5 ppm 40

EXAMPLE B Effect of Omphalotin on Meloidogyne incognita

[0276] Test nematode: Meloidogyne incognita

[0277] Test method:

[0278] The test substance is dissolved in methanol, pipetted into a 24well microtitre plate, and the solvent is evaporated. Subsequently, 400μl of nematode suspension are added into each well of the microtitreplate. When using Meloidogyne incognita, the number of nematodes is 100L2/400 μl. To enhance the solubility of the samples in aqueous medium,the plate is shaken at 100 rpm (IKA MTS 4 plate shaker) for 15 minutes,and the first microscopic control (Leitz inverse microscope) is carriedout subsequently. Evaluation is carried out after 16 h. Nematicidalactivity of omphalotin (μg/ml) Nematode LD 90 LD 50 Meloidogyneincognita 1-1.5 0.75

[0279] This test method is also particularly suitable for testing theactive compound content of omphalotin-containing fractions in thepreparation of omphalotin.

EXAMPLE C Effect of Omphalotin on Meloidogyne incognita in a Greenhouse(Test Plant: Lettuce)

[0280] Test nematode: Meloidogyne incognita

[0281] Test method:

[0282] The test substance, as a solution (10 ml per 250 ml of soil), isstirred homogeneously into a soil/leaf mould mixture, together withabout 700 nematodes (mixture of eggs and larvae). The treated andinoculated soil substrate is filled into pots of a size of 7×7×6 cm intowhich lettuce (variety Attraktion) is sown. The seeds are pressed intothe soil and covered with a thin layer of quartz sand. The pots areincubated in a greenhouse at 25° C. and kept uniformly moist. After 24days, evaluation is carried out by washing the roots and determining thenumber of galls formed per plant.

[0283] The efficacy of the test substance is 100% if no galls have beenformed, and it is 0% if the same number of galls has been formed as inthe control. Test substance (concentration based on the volume of soilin ppm = mg/l) Efficacy (in % Abbott) Omphalotin 0.5 ppm 48 Omphalotin2.5 ppm 96

EXAMPLE D Effect of Omphalotin on Meloidogyne incognita in a Greenhouse(Test Plant: Cucumbers)

[0284] Test nematode: Meloidogyne incognita

[0285] Test method:

[0286] Two cucumber plants (variety Bella) each are grown in a soil/leafmould mixture in pots of a size of 7×7×6 cm. The test substance isdissolved in methanol, and then diluted with water and put onto the14-day-old cucumber plants. Per pot, 10 ml of solution are used. 3 hoursafter the treatment, about 500 nematodes as a suspension are pipettedonto each pot. The pots are incubated in a greenhouse at a daytemperature of 28° C. and a night temperature of 20° C. Evaluation iscarried out after 20 days by washing the roots and determining thenumber of galls formed per plant.

[0287] The efficacy of the test substance is 100% if no galls have beenformed, and it is 0% if the same number of galls has been formed as inthe control. Test substance (concentration based on the volume of soilin ppm = mg/l) Efficacy (in % Abbott) Control methanol  0 Omphalotin 5ppm 61

EXAMPLE E Effect of the Substance According to the Invention onNematodes

[0288] Test nematode: Radophulus similis

[0289] Test method

[0290] The substance is dissolved in methanol. Dilutions are thenprepared and pipetted into the wells of a multiwell tissue culture plate(24 well). The methanol is then allowed to evaporate in a fume cupboard,and 0.8 ml of physiological sodium chloride solution is filled into thewells. 100 nematodes in 0.2 ml of water are added in each case by meansof a pipette.

[0291] After 24 hours of incubation at about 22° C., evaluation iscarried out under the microscope by determining the proportion of deadnematodes in the treated samples in comparison to the untreated control.

[0292] The efficacy of the test substance is 100% if all the nematodeshave been killed. If the same number of nematodes are alive as in thecontrol sample, the efficacy is 0%.

[0293] The following efficacies were found: Concentration of thesubstance according to the invention (ppm = mg/l) Efficacy in %(according to Abbott)  1 ppm 23 10 ppm 46 30 ppm 54

EXAMPLE F Effect of the Substance According to the Invention onNematodes

[0294] Test nematode: Pratylenchus penetrans

[0295] Test method

[0296] The substance is dissolved in methanol. Dilutions are thenprepared and pipetted into the wells of a multiwell tissue culture plate(24 well). The methanol is then allowed to evaporate in a fume cupboard,and 0.8 ml of physiological sodium chloride solution is filled into thewells. 100 nematodes in 0.2 ml of water are added in each case by meansof a pipette.

[0297] After 24 hours of incubation at about 22° C., evaluation iscarried out under the microscope by determining the proportion of deadnematodes in the treated samples in comparison to the untreated control.

[0298] The efficacy is 100% if all the nematodes have been killed. Ifthe same number of nematodes are alive as in the control sample, theefficacy is 0%.

[0299] The following efficacies were found: Concentration of thesubstance according Efficacy in % to the invention (ppm = mg/l)(according to Abbott) 10 ppm 67 30 ppm 97

EXAMPLE G Effect of Omphalotin on Insects

[0300] Test insect: Plutella xylosella

[0301] Test method:

[0302] The test substance is dissolved in methanol and diluted to thetest concentration using emulsifier-containing water (0.4 ml ofemulsifier W/l of H₂O). With the aid of a cork borer, discs of adiameter of 4.4 cm are punched from cabbage leaves. The leaf discs aredipped into the solutions of the test substances by means of a pair oftweezers and subsequently placed in Petri dishes (diameter 9 cm) linedwith filter paper (1 leaf disc/Petri dish). Each leaf disc is infectedwith 6 L3 larvae. Visual scoring is carried out after two, six and 16days. On the third and, if appropriate, on the sixth day of thetreatment, the food is replenished with untreated leaf discs.

[0303] The efficacy of the test substance is 100% if all test insectshave been killed, and it is 0% if the same number of insects are stillalive as in the control.

[0304] The following efficacies were found: Omphalotin concentrationEfficacy Efficacy (ppm = mg/l) (in % Abbott) (in % Abbott) after 2 daysafter 6 days 100 ppm 33 100

EXAMPLE H Effect of Omphalotin on Various Fungi and Bacteria

[0305] Test method:

[0306] Petri dishes are prepared using 10 ml of potato dextrose agar.The test substance is applied as a solution to the agar and spread withthe spatula until the solution has been soaked up by the culture medium.The inoculum of the microorganisms to be tested, as spores or mycelfragments, is stamped onto the agar plates by pressing a stamp, whichis, if appropriate, covered with felt, first onto a densely overgrownplate and then onto the test plate. After 5 to 6 days, the radial growthof the microorganism colonies is measured and compared to that of theuntreated control.

[0307] The efficacy of the test substance is 100% if no growth isdetected, and it is 0% if growth is identical to that of the control.Efficacy of omphalotin (10 ppm (= mg/l) based on the volumeMicroorganism of the culture medium) Fusarium culmorum 51 Pythiumultimum 25 Fusarium graminearum 23 Alternaria mali 70 Rhizoctonia solani90 Septoria nodorum 60 Phytophthora cactorum 50 Pseudocercosporella 55herpotrichoides Pyricularia oryzae 70 Xanthomonas versicatoria 95

[0308] This test method can also be used in a particularly advantageousmanner in the preparation of omphalotin to determine theomphalotin-containing fractions.

1. Organochemical compound (omphalotin) of the formula (I)

where—(terminal) represents —CH₃,

represents

or (terminal) represents

represents —CH₂—,

represents

and

represents


2. Organochemical compound (omphalotin) which is formed during thefermentation of Omphalotus olearius strains and which has the followingphysico-chemical data and parameters a) Appearance: white powder b)Solubility readily soluble in methanol, acetone and 2- propanol, lessreadily soluble in ethyl acetate and hardly soluble in cyclohexane andt-butyl methyl ether c) Molecular weight: 1317 d) Empirical formulaC₆₉H₁₁₅N₁₃O₁₂   (elemental   analysis): e) Spectra The data of the ¹HNMR spectra are shown in Tables 1 and 2 of the description.


3. Process for preparing the organochemical compound (omphalotin)according to claims 1 or 2, characterized in that suitablemicroorganisms of the genera Omphalotus and Lampteromyces, preferably ofthe genera Omphalotus, in particular Omphalotus olearius strains, arecultivated under aerobic conditions in a culture medium which containsassimilable carbon and nitrogen sources and mineral salts, and thedesired compound is isolated by customary methods.
 4. Process accordingto claim 3 , characterized in that the Omphalotus olearius strains No.83 039 (according to DSM 9737), 90 173 (according to DSM 9738), 91 050(according to DSM 9739), 92 095 (according to DSM 9740), 93 162(according to DSM 9741) or 90 170 (according to DSM 9742) and mutantsand variants thereof are employed.
 5. Organochemical compound accordingto claims 1 or 2, obtainable by the process according to claims 3 and 4.6. Pesticide, characterized in that it contains the organochemicalcompound according to any of claims 1, 2 and
 5. 7. The use of theorganochemical compound according to any of claims 1, 2 and 5 forcontrolling pests, in particular insects, nematodes and microbes. 8.Method for controlling pests, characterized in that the organochemicalcompound according to any of claims 1, 2 and 5 is allowed to act onpests, preferably on insects, nematodes and microbes, or their habitat.9. Process for preparing pesticides, characterized in that theorganochemical compound according to any of claims 1, 2 and 5 is mixedwith extenders and/or surfactants.
 10. Microorganisms of the generaOmphalotus and Lampteromyces, preferably of the genera Omphalotus, inparticular Omphalotus olearius strains, which produce the organochemicalcompound according to claims 1 or 2 during cultivation in a culturemedium containing carbon and nitrogen sources and mineral salts. 11.Omphalotus olearius strains No. 83 039 (according to DSM 9737), 90 173(according to DSM 9738), 91 050 (according to DSM 9739), 92 095(according to DSM 9740), 93 162 (according to DSM 9741) or 90 170(according to DSM 9742) and mutants and variants thereof are employed.12. Organochemical compound according to any of claims 1, 2 and 5 forcontrolling diseases which are caused by parasites, preferablynematodes, or microbes, preferably fungi and bacteria.